Ripe and unripe Cavendish bananas in slurry
form, used as substrate for fermentation, were prepared as
follows: A known weight of bananas (whole fruit including peels)
was chopped into small pieces and pureed with distilled water in
a blender to make a slurry with a banana-to-water ratio of 1:3.
The following supplements were added: (NH4)2SO4,
4 g/litre KH2PO4, 2 g/litre; MgSO4,
1 g/litre; and calcium pantothenate, 4.5 mg/litre. The pH of the
slurry was between 4.5 and 4.9.

The Organisms

Four strains of Aspergillus niger
(UPCC 3701, 3026, 3450, and 3809), two strains of Aspergillus
foetidus (UPCC 3702 and 3448), and mixed cultures of Endomycopsis
fibuligera (UPCC 2407) with Candida utilis (UPCC
2074) and of A. foetidus (UPCC 3448) with A. niger
(UPCC 3809) were used as test organisms. Fungi were maintained on
Ozapek Dox agar slants and yeasts on yeast malt agar slants at
29° to 30°C. These organisms were obtained from the culture
collection of the University of the Philippines Natural Research
Center.

Preparation of the Inoculum

Primary Inoculum

A.niger and A. foetidus were
cultivated on Ozapek Dox agar slants, while E. fibuligera and
C. utilis were cultivated on yeast malt agar slants. After
three or four days of incubation at 29 to 30 C, spore suspensions
of the organisms were transferred aseptically to bottles
containing the supplemented substrate solidified with agar. This
was done to acclimatize the organisms to the substrate. The
inoculated bottles were incubated at 29° to 30°C for three or
four days. The spore suspension prepared from these bottles was
used as the primary inoculum.

Secondary Inoculum

The secondary inoculum was prepared by adding 5
ml of the primary inoculum to 10 ml of sterile banana slurry in a
500-ml Erlenmeyer flask, which was then shaken for 24 hours.
After that, 60 ml of sterile medium was added, and shaking was
continued for another 24 hours. Then 80 ml of fresh, sterile
substrate was added, to make up a total volume of 150 ml, and the
flask was again shaken for 24 hours The resulting culture was
used as inoculum.

For succeeding fermentation runs, to minimize
the time lag between runs, the inoculum was prepared by
inoculating 150 ml of substrate with 10 to 15 ml of primary
inoculum and shaking it for 24 hours. For fermentations on a
larger scale, the inoculum was scaled up correspondingly.

Batch Culture Fermentation

Fermentation was carried out for 24 hours in a
5-litre reactor vessel (Marubishi Ltd., Japan) with a working
volume of 2.5 litres. The pH was maintained at between 4.0 and
5.0 by automatic addition of NH4OH. Air flow rate was
controlled from 0.5 to 5.0 litres/min, and agitation speed was
regulated from 200 to 600 rpm to maintain the dissolved oxygen
concentration above 1 ppm. Changes in pH, sugar concentration,
and biomass concentration were noted at regular intervals (every
four hours). Foam was controlled by the automatic addition of an
antifoaming agent, while temperature was maintained at
approximately 30°C.

Fermentation runs were carried out on a larger
scale in a 14-litre Microferm fermentor (New Brunswick Sci. Co.,
USA) with a working volume of 7 litres. It has automatic pH,
dissolved oxygen, temperature, and foam control. Maximum air flow
rate values of 16 litres/min and agitation speeds up to 1,000 rpm
can be attained. A 10 per cent v/v inoculum size was used.

Analytical Procedure

A 10 ml portion of every sample taken was
centrifuged. The supernatant was collected and analysed for sugar
content by means of the Somogyi-Nelson method of reducing. sugar
determination (35; 36). The volume of residue was noted, and this
was washed three times with 0.9 per cent saline solution and
dried to constant weight at 60° to 80°C in a vacuum oven in
aluminium foil boxes previously dried to constant weight. The
dried samples were analysed for their crude protein content by
the micro-Kjeldahl method (37).

Results and Discussion

The main pulp of the Cavendish banana contains
a considerable amount of carbohydrates, mostly starch and
reducing sugars, but is low in protein. The peels have a crude
fibre content of 2.08 per cent (unripe) and 1.93 per cent (ripe).
The extremely large reserves of polysaccharides make the banana
rejects a potential source of SCP.

Two methods were proposed for using the banana
rejects (fig. 2). The first involves treating the bananas with
dilute acid in an autoclave, which results in the breakdown of
starch and cellulosic materials into simple sugars. The
hydrolysate obtained is used as substrate for SCP production,
giving rise to a product consisting wholly of fungal mycelia or
yeast cells. The second method is the direct enzymatic
fermentation by selected fungi and yeasts of slurry prepared from
the bananas. The product obtained in this method consists of
yeast cells or fungal mycelia plus unhydrolysed banana residues.
All of the investigations carried out made use of the whole
banana fruit (pulp and peel), as it would not be economical if
only the pulp or the peels were used.

Acid Hydrolysis

Studies on the acid hydrolysis of bananas were
conducted. The effects of several factors, namely, acid
concentration, banana-to-water ratio, and time (duration) of
hydrolysis, on sugar yield were studied. Hydrolysis was carried
out at 121°C because results of studies done in our laboratory
on the acid hydrolysis of rice straw 138) as well as those
reported in the literature (24) showed that the yield of sugar at
this temperature was 30 per cent higher than the yield at 100°C
at all the acid concentrations used

Results show that maximum reducing-sugar yield
(as glucose) from dried, unripe bananas was obtained when banana
diluted with water to a 1:2 ratio was treated with 4 per cent H2SO4
for 30 minutes, or with 2 per cent H2SO4
for 1 hour at 121°C. Dried, ripe bananas had lower sugar yields
when subjected to the same conditions of hydrolysis. This is
because starch is converted to simpler sugars as the banana
ripens, and acid hydrolysis of the ripe banana results in further
degradation of these sugars to produce furfural and other
degradation products that could be inhibitory to microbial
growth. Hence, ripe bananas do not need to be hydrolysed in order
to prevent the production of toxic by-products. Much of the
reducing sugars present in ripe bananas can be directly used by
micro-organisms for SCP production.

TABLE 2. Fermentation Data on Some Yeasts and
Fungi Grown on Banana Slurry by the Batch Culture Method

Protein Content (N x
6.25) of Product (%)

Crude Crude Protein
Yield (g/ml) (%)

Conversion of Sub strafe
to Crude Protein

Aspergillus
niger (UPCC 3450)

ripe

19.25

0.0048

17.20

unripe

28.02

0.0065

15.82

A.
niger (UPCC 3026)

ripe

30.67

0.0076

32.90

unripe

19.88

0.0046

15.23

A.
niger (UPCC 3701)

ripe

27.51

0.0085

40.43

unripe

22.25

0.0054

21.01

A.
niger (UPCC 3809)

ripe

25.31

0.0065

26.29

unripe

23.85

0.0066

19.41

A.
foetidus (UPCC 3448)

ripe

27.83

0.0083

45.11

unripe

20.88

0.0055

14.82

A.
foetidus (UPCC 3702)

ripe

26.58

0.0063

31.50

unripe

21.75

0.0050

13.89

Endomycopsis
fibuligera

(UPCC 2407)
and Candida

ripe

30.08

0.0070

24.05

utilis (UPCC
2074)

unripe

30.26

0.0076

16.02

A. foetidus
(UPCC 3448)

ripe

41.30

0.0082

57.85

and A. niger
(UPCC 3809)

unripe

32.09

0. 0080

31.36

a. Average values of several fermentation runs.

Studies on acid hydrolysis were later abandoned
because this process does not appear to be economically feasible.
Considering the amount of acid, heat, water, and time required
for the process, this method is an expensive step in the
production of SCP. Thus, the emphasis in the present research
project is confined to direct enzymatic fermentation by
micro-organisms.

One phase of this research that is being
investigated is concurrent enzymatic starch hydrolysis and yeast
cell multiplication through the use of a mixed culture. This
process would be cheaper than starch hydrolysis by acid followed
by yeast propagation and is accomplished by inoculating the
banana slurry with equal volumes of Endomycopsis fibuligera,
a mycelial yeast that produces an amylase capable of breaking
down starch into glucose, and Candida utilis, a known
food yeast that cannot use starch directly but can use glucose.

Growth of these organisms on unripe banana
substrate increased the crude protein content of the final
product (yeast cells and unhydrolysed banana residues) to an
average value of 30.26 per cent. On ripe bananas, average final
crude protein content was 30.08 per cent. The fermentation data
are shown in table 2. The relevant equations are:

During fermentation, considerable weight loss
is observed with unripe banana substrate, while there is a slight
decrease in dry weight when ripe bananas are used.

Dry weight measurements, however, cannot be
used to follow the growth of the organisms since the samples are
a mixture of microbial cells and unhydrolysed banana residues
that cannot be separated. The behaviour of dry weight
measurements can be interpreted in terms of a balance between
microbial growth, which tends to increase dry weight, and
hydrolysis of the banana slurry, which tends to decrease dry
weight of the product, The final product is lower in dry weight
because of incomplete conversion to microbial cells of the
hydrolysed banana substrate. Part of it is metabolized to other
products such as CO2 and residual sugars or is
converted to energy (as ATP), which is required for hydrolysis.

On both ripe and unripe bananas, reducing-sugar
concentration in the supernatant remains very low (less than
0.003 g/ml) throughout the fermentation period (fig. 3). This is
because C. utilis immediately utilizes the sugars
released by E. fibuligera from the banana substrate.

Two other organisms used mixed culture are A.
niger 3809 and A. foetidus 3448. A. niger
is known to produce large amounts of amylases and also
cellulases; A. foetidus produces only small amounts of
amylases; and both can use glucose equally well. In contrast to
the mixed culture of two yeasts, reducing sugar is found to
accumulate in the medium. When unripe bananas are used, rapid
enzymatic hydrolysis occurs during the initial growth phase (fig.
4B). Sugar concentration increases rapidly during the first 12
hours (0.0002 to 0.0167 g/ml, or an 80-fold increase), which is
accompanied by a sharp decline in dry weight. Dry weight then
increases with time with a corresponding decrease in sugar
concentration.

In the case of ripe bananas, despite the high
concentration of sugar initially present (0.0242 9/ml), slight
hydrolysis still occurs during the first eight hours, causing the
sugar concentration to increase to 0.0292 g/ml with a
corresponding decrease in dry weight (fig. 4A). This may mean
that the enzymes are not inhibited at this sugar level. Rapid
utilization of the sugar follows, accompanied by an increase in
dry weight. Accumulation of sugar in the medium is not
surprising, as both organisms are amylase-producing.

The second phase under investigation is the
direct fermentation of bananas into microbial protein by one
organism alone. Different isolates of A. niger and A. foetidus
were used in this study. Results are shown in table 2.

When A. niger 3809 is used alone,
final dry weight is lower for both ripe and unripe bananas,
although the same changes in dry weight during fermentation are
observed as in the mixed culture of the two fungi (fig. 5A and
B). The same general behaviour of reducing sugar in the medium is
also observed, With ripe bananas, two peaks are observed, one at
4 and the other at 12 hours. However, the increase in sugar
concentration is small (0.0355 to 0.0412 and 0.0380 g/ml). In the
case of unripe bananas, the peak is reached 8 hours after
inoculation (0.0052 to 0.0165 g/ml, representing a threefold
increase in sugar concentration), with a smaller peak at 16 hours
(0.0093 g/ml).

When A. foetidus 3448 is used alone on
ripe bananas, a very slight peak in sugar concentration appears
after 4 hours (0.0178 to 0.0201 g/ml) accompanied by a slight
decrease in dry weight (fig. 5C and D). In the case of unripe
bananas, final dry weight is lower, with the dry weight changing
gradually during the fermentation, as shown by a smooth, rounded
curve, and the peak in sugar concentration occurring only after
20 hours (0.0053 to 0.0101 g/mg, or a twofold increase), which
probably indicates slow hydrolysis of the banana. This confirms
an earlier statement that the amylase of A. foetidus
3448 is weaker than that of A. niger 3809.

We can conclude that the use of A. foetidus
3448 and A. niger 3809 in combination is better than
using either one alone, because the crude protein content of the
final product and the efficiency of conversion of substrate to
protein is higher when the mixed culture is used, whether with
ripe or unripe bananas.

A. foetidus 3702 behaves in a similar
manner to A. foetidus 3448 on both ripe and unripe
bananas (fig. 5E and F). There is very slight hydrolysis of the
substrate, indicated by the absence of sharp peaks in
reducing-sugar concentration in the medium. A. foetidus
3448 may even be slightly better than A. foetidus 3702
because more sugar accumulates in the medium when the former is
used. It has also been observed that the crude protein content of
the products and conversion efficiency of the two organisms are
similar,

When the three remaining strains of A.
niger (3701, 3450, and 3026) are compared, final dry weight
is higher than the initial weight on ripe bananas for A.
niger 3701, lower for A. niger 3450, and
approximately the same for A. niger 3026 (fig. 6). A.
niger 3026 produces the highest peak in reducingsugar
concentration after 8 hours (0.0465 to 0.2789 g/ml, for a sixfold
increase), while the dry weight hardly changes. For A. niger
3701, a small peak occurs at 8 hours, with the dry weight
surprisingly reaching a peak at this point also. This may be
attributed to sampling error With A. niger 3450, a rapid
decline in sugar concentration prior to the occurrence of a peak
that is lower than the initial value is accompanied by a decrease
in dry weight.

Two peaks in sugar concentration are always
observed, a large peak during the initial growth phase and a
smaller peak after most of the sugar has been used up. This may
be attributed to increased activity of the starch-hydrolysing
enzymes when the sugar concentration drops to a low level or to
decreased growth rate of an organism that has probably reached
the stationary phase while enzyme activity remains the same,
leading to accumulation of the released sugar in the medium. This
is not observed with A. niger 3809.

On unripe bananas, the final dry weight is
lower for all three organisms. No appreciable peaks in sugar
concentration are observed, indicating that the hydrolysis taking
place is just enough to support microbial growth. A. niger
3809 may have stronger amylase activity on this substrate, since
more sugar is released Into the medium (0.0052 to 0.165 g/ml).

When the crude protein content of the final
product and conversion efficiency are compared, growth on ripe
bananas generally produces higher values than on unripe bananas. A.
niger 3701 has the highest average crude protein content on
ripe bananas (27.51 per cent). The value for A. niger
3026 was taken from one fermentation run only and so cannot be
interpreted as the highest. A. niger 3701 also has the
highest conversion efficiency. On unripe bananas, A. niger
3450 has the highest protein content, but A. niger 3701
has the highest conversion efficiency.

For all the fermentation runs performed, the
final product is almost always lower in dry weight than the
starting material when unripe banana slurry is used as substrate.
In some cases, reduction in weight of more than 50 per cent is
observed. In the case of ripe bananas, final dry weight may be
higher or lower. In contrast to the mixed culture of yeasts,
accumulation of reducing sugar in the supernatant is always
observed sometime during the fermentation when fungi are used.
This drops to low levels as the fermentation continues.

A mixed culture of A. foetidus 3448
with A. niger 3809 appears to be the best on ripe and
unripe bananas, followed by a single culture of A. niger
3701 and a mixed culture of E. fibuligera 2047 with C.
utilis 2074. However, definite conclusions cannot be drawn
until a more direct measurement of starch utilization has been
carried out. A method of starch analysis is being studied, and we
hope analyses can be completed in the near future. An indirect
method of following microbial growth - analysis of the protein
content during fermentation - is also being studied. Relative
nutritive values of the different types of microbial protein
produced cannot be compared at present because analyses of the
amino acid contents and toxicological tests have yet to be
carried out.

Results of this research so far have
demonstrated the possibility of growing fungi and yeasts on
banana waste with relatively high crude protein content. However,
it is not comparable to that of current protein sources such as
plant proteins and commercial animal feeds (e.g., soy meal, 45 to
50 per cent; fish meal 60 to 65 per cent). It has also been shown
that no preliminary chemical treatment is necessary as long as
the appropriate organisms are used. Further studies will be
conducted in an attempt to increase the protein content and
conversion efficiency.